Bacterial fermentation is the most efficient industrial process for manufacturing biological molecules, and has been the method of choice for bio-therapeutics production. In the past few years, however, drug manufacturers have begun to face up to the problems associated with batch and fed-batch fermentation, in which the whole process takes place in a single vessel over a period of a week or more yielding only one fermentation vessel volume of fermentate. Cells are grown to relatively high density (˜OD=100) and then induced to make the product for a relatively short period (hours) before they start to die. Batch processes are vulnerable to failure, many due to genetic instability of the bacterial strains used, which are often metabolically inefficient and highly subject to stress under induction. Metabolic stress can induce responses in the cells which can compromise product formation and integrity and damage the genome, slow growth or even kill cells. Mutants that no longer make product can quickly overtake the culture or bacteriophage or lysogens from the genome can kill or lyse the entire culture.
A more efficient and reliable fermentation protocol is a process where nutrients are continuously added to the culture as needed and bacteria and products are harvested continuously. Such continuous flow bioreactor systems may be operated as a chemostat, in which the volume of the bioreactor is held constant by synchronizing the input of nutrient medium to the outflow of cells and spent media (the dilution rate) to produce a physiological steady state in the resident bacteria. In commercial application this steady state is ideally set to hold the bacterial cells at the physiological optimum point of maximum product formation. Unfortunately, chemostat operations are highly sensitive to mechanical disruption since the delicate balance established by the dilution rate can be upset by physical changes to flow through the system. Furthermore, genetic changes in the population within the bioreactor can also upset the physiologic steady state or nonproductive mutants can take over the culture [See van Heerdon and Nicol, Microbial Cell Factories 12:80 (2013)].
In addition to the mechanical and genetic challenges inherent in continuous flow bioreactor operations, there are difficulties associated with establishing a steady state in traditional single vessel chemostats with recombinant systems requiring induction of gene expression to produce the product of interest. Typically, such a system is grown in an initial batch or fed batch phase to a predetermined optimal cell density and then exogenous inducer such as IPTG is added and the chemostat mode then established using a feed input containing properly diluted inducer to maintain the level of induction throughout the course of the fermentation. Not only is this technically difficult, but frequently, bacteria that undergo mutational changes that lessen the burden of induced gene expression tend to overgrow the population within the bioreactor resulting in a loss of productivity. Such problems also occur in batch fermentations, however the impact on productivity is generally less significant since the batch or fed batch fermentation has a finite (and generally short) lifetime.
More recently, a continuous flow fermentation system was described in which two-vessels were combined in a process to produce covalently closed circular DNA plasmids by induction of a temperature sensitive origin of replication (U.S. Patent Publication No. 2008/0318283). In this process the cells in the first vessel were grown at a relatively low temperature resulting in low plasmid copy number within the cells. Under this condition the desired cells containing the highly inducible plasmid and undesired cells comprising mutations that decrease overall plasmid copy number have little or no growth advantage between themselves. In this system only upon passage into the higher temperature second vessel is plasmid copy number induced and a selective advantage conferred upon the undesired cells containing plasmids with lower induced copy number. Thus, the first (seed) vessel serves as a continuous inoculum for the second (production) vessel. Continuous inoculation with the desired cells limits the ability of undesired faster growing mutants becoming established in the production system, since the mutants are subject not only to continuous washout, but to continuous replacement by uncontaminated inoculum.
Although this presents an elegant solution to part of the problem of production cell stability it is only a partial solution. Direct cellular engineering to improve production strain stability can be coupled with the two-vessel system to improve the overall process. Further, it is not entirely certain that induction schemes involving chemical or biological induction, rather than thermal induction, will prove equally useful and they will, of necessity require more complex input streams to provide a continuous level of inducer within the production vessel while keeping the seed vessel free from inducer. In addition, a two-vessel system may provide an ideal framework for determining optimum production conditions by allowing serial variation of different conditions with a uniform cell source. In this case, the production vessel is held at the desired condition for the required period of time to establish the steady state, samples are taken for productivity determination and the next experimental condition established merely by modifying the feed or other experimental parameter (oxygen tension, pH, amount of inducer, etc.) and allowing the system to come to the new steady state before taking a new round of samples. Such a process can be repeated any number of times without requiring any input from the user other than providing the new experimental inputs. Much of this can be done entirely by preprogrammed computer controllers. Further, a single seed vessel can serve multiple production vessels facilitating parallel experimental or production operations.
Using an appropriate apparatus, referred to here as the “C-flow” apparatus and specially engineered bacteria, continuous processes use less energy, manpower, downtime, capital equipment outlay and footprint, and when set up correctly can run for months, producing on the order of a vessel volume of fermentate per day, at cell densities and product concentrations more than double that of a fed batch process for the same product. With the C-flow system a vessel of a given size can produce from 5 to 50 times more product than a comparably sized vessel using a fed batch system within a period as short as three weeks.